This is in the general area of genetic engineering of nucleic acid sequences, especially RNA sequences, and compositions and diagnostics for use therein.
Discoveries in the basic realm of molecular biology over the past five years have led to the realization that RNA has a series of distinct capabilities and biological activities previously unsuspected. The most important of these novel RNA-level discoveries has been the finding that RNA can be an enzyme as well as an information carrier.
Since 1982, several unexpected diseases caused by RNA-based pathogenic agents have emerged. These include the lethal Acquired Immune Deficiency Syndrome (AIDS) and delta hepatitis, a particularly virulent form of fulminant hepatitis caused by a viroid-like RNA agent. These blood-borne diseases are spread at the RNA level, manifest themselves in cells of patients, and are by now present within the bloodstream of millions of individuals.
Conventional biotechnology, with its reliance on recombinant DNA methods and DNA-level intervention schemes, has been slow to provide valid approaches to combat these diseases.
As described by Thomas R. Cech, in JAMA 260(20), 3030-3034 (November 1988), certain RNA molecules can mediate their own cleavage or splicing or act as enzymes to promote reactions on substrate RNA molecules, and can therefore play an active role in directing cellular biochemistry. These findings suggest the possibility that other cellular RNAs, including the RNA components of small nuclear ribonucleoproteins, of the ribosome, and of various ribonucleoprotein enzymes, are catalysts. These activities are emerging as important contributors to the understanding of RNA processing. Splicing of introns and exons in mRNA, first described in 1977, is a major part of the RNA processing field. In the decade since RNA splicing was first described, four major categories of introns have been found, each with its own RNA splicing mechanism. As with many other biological reactions, RNA splicing requires catalysts to speed up the rate of the reaction and to ensure that splice sites are chosen accurately. In at least four systems described to date, RNA catalysts, or ribozymes, appear to be involved in RNA cleaving and splicing reactions. Ribozymes are defined as specific domains of RNA molecules which have enzymatic activity - either acting as an enzyme on other molecules, or undergoing intramolecular catalysis in reactions such as self-splicing or self-cleaving.
Ribozymes can be used as sequence-specific RNA cleavage agents in vitro, providing useful tools for biochemical studies of RNA. In studies of the rRNA precursor of Tetrahymena thermophila, a ciliated protozoan, Cech found splicing to be catalyzed by the folded structure of the intron itself. The observation of self-splicing was later extended to related group I introns and to the structurally distinct group II introns. In some cases the RNA catalyzed reactions also involve proteins. Splicing of group I introns takes place through two transesterification reactions, exchanges of phosphate esters that leave the total number of phosphodiester bonds unchanged. In the first transesterification step, the 5' splice site is cleaved as guanosine is added to the 5' end of the intron. In the second step, the 3' splice site is cleaved as the exons are joined. To help catalyze these reactions, the intron provides binding sites for the exogenous guanosine and for nucleotides near its own splice sites. In addition, the intron provides an active site that facilitates the reaction.
A structurally distinct group of introns found in fungal mitochondria, the group II introns, described by Michel, et al., EMBO J. 2, 33-38 (1983), also undergoes self-splicing. As with the group I introns, splicing occurs through two transesterification reactions, the first involving the 5' splice site and the second the 3' splice site. In contrast to the group I intron mechanism, the attacking group (nucleophile) for the first transesterification is the 2'-hydroxyl group of an adenosine located within the intron. The product of transesterification is a branched RNA called a lariat. Nuclear mRNA introns in all eukaryotes studied appear to undergo splicing in the same manner as the group II introns.
An enzyme can be defined as a molecule that greatly accelerates the rate of a chemical reaction, with great specificity for its substrates and for the type of reaction it facilitates, which is not consumed in each reaction, so that one enzyme molecule can interact with numerous substrate molecules. Self-splicing RNA of the type present in group I and group II introns is not a true enzyme since it is altered by the reaction. As described by Cech, et al., in PCT/US87/03161, however, deletion or substitution of some of the nucleotides can be used to convert the self-splicing group I intron into an enzyme having a specificity for a sequence of four nucleotides. Removal of the first 19 nucleotides of the Tetrahymena rRNA intron produces a form called the L-19 intervening sequence RNA. This molecule no longer contains sites for intramolecular reactions but can mediate reactions on added substrate RNAs without itself undergoing any net change in the process. The first activity described for this RNA enzyme was the assembly of short chains of RNA into longer chains, i.e., an RNA polymerization activity, as reported by Zaug, et al., Science 231, 470-475 (1986).
A second activity of the RNA enzyme derived from the Tetrahymena intervening sequence is that of a sequence-specific endoribonuclease, described by Zaug, et al., Nature 324, 429-433 (1986). This RNA enzyme is able to cleave other RNA molecules that are single stranded with considerably more sequence specificity than any known protein ribonuclease (RNase). Recognition is provided by the sequence of nucleotides preceding the cleavage site, and cleavage is accomplished by guanosine addition. Site-specific mutagenesis of the enzyme active site (the sequence 5'-GGAGGG-3') alters the cleavage specificity in a predictable manner so that it has been possible to synthesize endoribonucleases to cut at a variety of sequences.
Two other major categories of RNA catalysis have been investigated, the first including examples from several different organisms of RNase P, a ribonucleoprotein enzyme that makes a specific cleavage in tRNA precursors; and the second including a variety of self-replicating RNAs that infect plants and animals, the viroid-like pathogens (or VLPs). As reported by Guerrier-Takada, et al., Cell 35, 849-857 (1983), the reaction involving the RNase P is similar to that catalyzed by the group I and group II introns, with the difference that the attacking entity is water instead of a ribose hydroxyl group.
Several of the VLP RNAs that infect plants, including one viroid (a small circular RNA that is infectious by itself) and several virusoids and satellite RNAs (circular and linear RNAs, respectively, that are encapsulated by the coat proteins of certain plant RNA viruses), undergo efficient site-specific self-cleavage in vitro, described by Prody, et al., Science 231, 1577-1580 (1986), and Hutchins, et al., Nucleic Acids Res. 14, 3627-3640 (1986). These RNAs share a small structural domain, consisting of only about 30 nucleotides, called a "hammerhead". During infection, self-cleavage is thought to be responsible for conversion of linear RNA multimers produced by rolling circle replication into unit-size progeny. The requirements for cleavage, which leaves a cyclic phosphate at the 3' end of the upstream cleavage product, are described by Uhlenbeck, Nature 328, 596-600 (1987) and Forster, et al., Cell 50, 9-16 (1987).
Hepatitis B virus, having a small, circular, partially double-stranded DNA genome, causes viral hepatitis and hepatocellular carcinoma in man. Hepatitis delta virus, containing a small, circular RNA, is a satellite virus of the hepatitis B virus. Superinfection of carriers of hepatitis B virus with hepatitis delta virus causes a fulminating hepatitis frequently leading to death. The hepatitis delta virus RNA is circular and has a structure characterized by intramolecular base pairing, similar to that of viroids and virusoids that infect plants, as demonstrated by Wang, et al., Nature 323, 508-514 (1986), and Chem, et al., Proc.Natl.Acad.Sci.USA 83, 8774-8778 (1986). Sharmeen, et al, demonstrated in J. Virol. 62, 2674-2679 (1988), that hepatitis delta virus RNA can undergo a type of self-cleavage in vitro. The cleavage products have 2',3'-cyclic phosphate and 5'-hydroxyl termini. The sequences and structures responsible for self-cleavage were not delineated but were predicted to be different from the hammerhead motif found in virusoids.
In a related finding Branch, et al., in Science 243, 649-652 (Feb. 3, 1989), demonstrated the existence of a novel structural element in HDV genomic RNA which lies within the highly conserved domain of HDV RNA and may be related to the local tertiary structure previously mapped to the central conserved region of the plant viroid genome. These authors also pointed out the close proximity of this structural element to the site of delta RNA self-cleavage. At the same time, Wu and Lai reported in Science 243, 652-655 (Feb. 3, 1989), that a 148 nucleotide subfragment of hepatitis delta virus RNA reversibly undergoes cleavage and ligation. The direction of the reaction is determined by the presence or absence of Mg.sup.2+ ions, with the presence of Mg.sup.2+ favoring the cleavage reaction. Ligation requires specific conformation of the RNA molecules involved and occurs only between two cleaved RNA fragments that are held together by hydrogen bonds. Ligation occurs on removal of Mg.sup.2+ by EDTA. In March 1989, Wu, et al., reported in Proc. Natl. Acad. Sci. USA 86, 1831-1835, that cleavage can be accomplished with subfragments of the hepatitis delta virus 1.7 kb genome as short as 133 nucleotides in the presence of at least 500 .mu.M Mg.sup.2+ or Ca.sup.2+, much lower concentrations than are required for cleavage by hammerheads, at a pH from 5.0 to 9.1, generating a 5' fragment with a terminal uridyl 2',3'-cyclic monophosphate residue and a 3+ fragment with a guanosyl residue with a 5'-hydroxyl group.
There have been a number of suggestions in the literature that ribozymes may have utility as reagents or as therapeutic agents, although little has been accomplished in implementing this goal. The Tetrahymena sequence, as well as the subsequently discovered sequence in yeast, is not a true enzyme since it is not regenerated in the process but instead acts in a stoichiometric ratio. Although it is possible to engineer fragments of this sequence which have enzymatic activity and are able to cleave and ligate RNA, a disadvantage to these fragments is that they are very large (requiring more than 200 residues of the original 415 nucleotide sequence) and of limited specificity. It has been suggested that the RNA subunit of E.coli RNAse P (MI RNA) which cleaves extra RNA sequences from the 5' ends of tRNA precursors, to create mature tRNA molecules can be engineered so that its substrate-recognition region is twenty nucleotides or less.
The viroid-like pathogens, VLPs, can be divided into two classes: Class I, free living viroids; and Class II, including virusoids and satellite viroids (RNA molecules which require a helper virus to replicate). The hepatitis delta virus is a Class II VLP by this definition. VLPs have two types of ribozymes. The first of these types, the "hammerhead", is being commercially exploited by Haseloff and Gerlach of CSIRO, Canberra, Australia. Uhlenbeck, Nature (1987), first developed these small (down to 18 nucleotides), and relatively specific ribozyme sequences from plant viroids such as avocado sunblotch viroid and the satellite RNAs of tobacco ringspot virus and lucerne transient streak virus. In their present forms, the Tetrahymena ribozymes have four-base recognition sequences and the hammerhead ribozymes have approximately 12-base recognition sequences. Any four-base sequence appears several times, on average, in an RNA the size of a typical mRNA, in contrast to 12-base sequences, allowing these ribozymes to be used in a complementary fashion to cleave RNA.
As far is known, none of these ribozymes has been used so far for anything other than as laboratory reagents for cleaving and splicing of RNA with somewhat limited specificity. In addition to the limitations of size, the relatively broad specificity of the ribozymes has made it difficult to target only the RNA to be cleaved, especially without doing any damage to the host RNA. No non-plant viroids besides hepatitis delta virus have been definitively characterized at this time. Thus, the key knowledge for harnessing any class of ribozyme, including the delta ribozyme, i.e., knowledge of its detailed, primary, secondary, and tertiary structure resulting in understanding of its mechanism, has yet to be achieved.
It is therefore an object of the present invention to provide methods and compositions for specifically cleaving targeted RNA sequences.
It is a further object of the present invention to provide methods and compositions for specifically cleaving RNA, both in vitro and in vivo, for the treatment of disease conditions which involve RNA expression, such as AIDS.
It is also an object of the present invention to provide methods and compositions for detecting, and treating, the viroid-like agent causing delta hepatitis, as well as additional viroid-like agents, including the agent responsible for Crohn's disease.
It is yet another object of the present invention to provide methods and compositions for constructing tissue specific expression vectors and vaccines based on the delta hepatitis viroid.